research to routine workflows for large and small molecules using...

Research to Routine Workflows for Large and Small Molecules using the Q Exactive HR/MS

Sept. 22, 2011

Patrick BennettDirector Pharma Strategic MarketingThermo Fisher Scientific

2

Agenda• Definitions

• Research to Routine• Small Vs. Large Molecule• Physicochemical• Types of instruments/technology and experiments• Small molecule regulations vs Large molecule regulations

• Application of HR/MS• Why HR/MS• Q Exactive

• Proven Proteomic workflow tools applied to routine quantitation

3

Definitions

• Pharma – “traditional” pharmaceutical company – focused primarily on small molecule drug discovery, development and sales. May have large molecule components and includes CRO’s.

• BioPharma – can be same company as Pharma or a company that specifically focuses on biologics based therapeutic agents. Includes CRO’s.

• Biologics – complex, natural proteins, blood products, clotting factors, antibodies, mAb, peptides, analogues, truncated forms, fusion proteins, non-protein macromolecules RNA/DNA, antisense RNA, aptamer’s/Oligonucleotides

• Large molecule – biologics; based on molecular weight

• Small molecule – traditional drugs; based on molecular weight.

4

RESEARCH TO ROUTINE

5

Biologics Development: Research to Routine

Research Discovery DMPK QC/QA

Unregulated Regulated•Range of traditional “Proteomics”experiments through to high throughput screening.•Research focus, minor component interest, many analytes, same type of test. •Internal based procedures rather than regulated SOP’s

•Routine and Semi-Routine experiments supporting safety and efficacy studies. •Few drugs, many samples, high expense•GLP & GMP environment – strict SOP’s

Activ

ities Traditional

Proteomics,Protein-protein interactionsPathway analysisActivity, PD Biomarker dev.

PTM AnalysisDisulfide MappingProtein CharacterizationStructural AnalysisImmunogenicityNeutralization

PK/PD, LBA’s, Cell based assays, PTM Optimization,Protein Characterization, Safety, Efficacy, Metabolism, DDI

PK/PD, CQA/QbD, Production Characterizations,Drug monitoring

6

Small vs Large Chemical Characteristics

Characteristic Small LargeMolecular Weight <800 >5000Endogenous No Often YesSolubility Hydrophobic HydrophilicPurity Homogeneous HeterogeneousImmunogenic No YesConjugated Yes NoValence Monovalent DivalentKinetics Fast SlowDetection Isotopic Activity/functionalStability Chemical/enzymatic Immunologic/enzymaticMetabolic interference Yes/knowledge UnknownProtein Binding Yes No

7

Small vs Large Analytical Methodology

Characteristic Small LargeBasis of Measurement Analyte Antigen-Ab reactionDetection Direct IndirectReagents Common and available Unique, not commercial

or availableAnalytes Small Small and macromoleculeSample Preparation Yes NoCalibration Curves Linear Non-LinearAssay Environment Organic AqueousDevelopment Time Weeks MonthsTechnology LC/MS (Affinity based

Extraction)LBA: ELISA, RIA, ECL, Multiplexing

Stability Drug Drug + ReagentsRegulations Very defined and rigid Some flexibility

8

APPLICATION OF HR/MS

9

Specificity = Resolution + Mass Accuracy

Resolution: 10k, 30k, 50k, 100k

279.12 279.14 279.16 279.18 279.20m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

Ethinyl-Estradiol, 279.17434

Butyl-Phthalate, 279.15909 (ubiquitous background ion)

54 ppmapart

10

492.2665

492.2495

492.2634

R: 35K R: 140K

m/z

492.2661

492.2511

R: 70K

30ppm

High Resolution Ensures Accurate Target Selection

• Accurate quantification is only possible when the target can becorrectly identified.

• An interference which is 30 ppm apart from the target can becompletely separated using high-resolution of 140,000 using theQ Exactive.

11

When does HR/MS make sense?

• Research phase• Early to late discovery phases• Pre-clinical phase• QA/QC - characterization• Ligand binding assay development

• Specificity testing• Stability testing

• Assay troubleshooting, unexpected results• Reagents unavailable, difficult to produce, expensive• Bridging methods, reagents• Biomarker research, multiple biomarkers

12

RoutineHigh throughput

Optimized assays

Pharma &Biopharma

Quantitation, Leachables, Extractables,

Impurities, QA/QC

DevelopmentMedium throughput

Verification

Translational Research,

Biopharma,Metabolomics,

MetabolismDrug Discovery,

Various Biomarker

ResearchLow throughput

Discovery

TraditionalProteomics,

Metabolomics, Metabolism,

Biomarker Research

Research to Routine: Range of Experiments for LC/MS

All Q TOF All Q TOF Triples &Q Trap

QuantitativeQualitative ▪ Identification ▪ Confirmation ▪

Q ExactiveOrbitraps Triples

13

Q Exactive TM Hardware Innovations

• S-Lens ion source• Quadrupole mass filter• Advanced signal processing

14

Specifications/Details

• Thermo Scientific HyperQuad mass filter• Mass range: 50-4000 m/z• Linear range: 4-5 orders of magnitude• Variable precursor isolation width selection from 0.4 Da to full mass

range• Resolution : up to 140,000

• 17k, 35k, 70k, 140k at m/z 200• scan speed dependent on resolution setting

• Sources:• ESI probe compatible with liquid flow rates of < 1 μL/min to 1 mL/min

without splitting • APCI source compatible with liquid flow rates of 50 μL/min to 2 mL/min

without splitting • Nanospray/microspray

15

• Sensitivity gain 5 – 10 x with SIM mode

• The gain will be higher in more complex matrices

What Do We Gain by Selected Ion Monitoring?

• In Full MS, total C-trap chargecapacity is shared between multiplesignals of different intensity

• Signal-to-noise ratio becomesdependent on the ratio of compoundof interest to other analytes-muchless so in SIM!

• In Orbitrap instruments, SIM couldbecome MRM without any additionaloverhead!

0

20

40

60

80

100

0

20

40

60

80

100

195.0876N=248402.81

195.0877N=20741.58

NL: 1.94E8[150.00-2000.00]

NL: 1.12E8[190.10-200.10]

Full MS

SIM (10amu)

S/N = 745IT= 0.245 ms

For the same target:S/N = 5400IT= 1.321 ms

Lowest signal250330

Lowest signal28240

0

1000

2000

3000

4000

5000

6000

195.082 195.084 195.086 195.088 195.09 195.092 195.094

S/N

(spe

ctru

m)

S/N (FMS) S/N (SIM10)

Gain in sensitivity (7x)

Caffeine

16

QUANTITATION

17

Traditional Workflow (Triple Quadrupoles)

NCEs

TuneMRM

PP Sample Clean Up

Bioanalysis

PK Estimates (NCE only)

Tuning MRM takes time & requires some level of expertise

MRM methods are not easily transferable between platforms from different vendors, which makes scalability difficult.

Peptides require determination of charge state and the optimal SRM transition, which is again platform dependent. Expertise required, even for a basic assay.

Limits the number of transitions (duty cycle & no. of scans per analyte).

Difficult to automate set-up to get sequence information – expertise required.

LC-SRM Analysis

18

HRMS Workflow (Q Exactive)

No compound dependent tuning required –easier to use/faster to set-up

Post-acquisition data analysis

Providing PK data as well as critical new information (metabolites, biomarkers)

More value in terms of the Fail Fast paradigm

LC-HRMSNCEs

PP Sample Clean Up

Bioanalysis

PK Estimates (NCE)

PK Estimates Metabolites

Multiple Analytes (IS)

Peptides

Post-Acquisition Data Query

19

Quantitation Summary of Small Molecules on Q-Exactive

Q ExactiveFull Scan

Q ExactiveTargeted-SIM

Triple Quad Vantage (SRM)

Q-Exactive achieves similar quantitation performance of above compound mixture as Vantage

Compound LOQ Curve Fit

Oxycodone 50 fg L - 1/X2Paroxetine 100 fg Q - 1/X2

Ketoconazole 50 fg L - 1/X2Clonazepam 500 fg Q - 1/X2Verapamil 50 fg Q - 1/X2

Clopidogrel 50 fg L - 1/X2

LOQCurve

Fit50 fg L - 1/X250 fg L - 1/X250 fg L - 1/X2

500 fg Q - 1/X2

50 fg L - 1/X2

50 fg L - 1/X2

LOQ Curve Fit

50 fg L - 1/X250 fg L - 1/X2

1000 fg Q - 1/X2100 fg L - 1/X2

10 fg Q - 1/X2

50 fg Q - 1/X2

20

Uroguanylin in Glucagon Peptide Matrix (Full Scan) – Q Exactive

Test-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.17E6T: FTMS + p ESI Full ms [820.00-1700.00]

850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

871.66876

1161.88527

981.82791

1667.61833

1177.54283

834.31346

939.13037 1026.997631472.23880

891.16079

1128.20213 1195.51589 1689.600491408.69556 1529.258531266.47914 1356.68300 1607.28170

[M+H]+[M+2H]2+

21

Uroguanylin Singly Charged SpeciesTest-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.32E5T: FTMS + p ESI Full ms [820.00-1700.00]

1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

1667.61833R=25584

z=1

1668.62129R=25350

z=1

1669.62036R=25986

z=1

1670.62126R=26333

z=1

1671.62188R=26133

z=11672.62227

R=25043z=1

1673.62198R=26044

z=1

Uroguanylin was quantified by summing the first 3 isotopes of both singly and doubly charged ions.

22

Test-003 #487-492 RT: 2.28-2.30 AV: 6 NL: 1.13E5T: FTMS + p ESI Full ms [820.00-1700.00]

833.5 834.0 834.5 835.0 835.5 836.0 836.5 837.0 837.5 838.0m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e A

bund

ance

834.31346R=36727

z=2

834.81445R=35417

z=2

835.31439R=36214

z=2

835.81451R=36408

z=2

836.31443R=35710

z=2836.81542R=32095

z=2

837.40331R=33958

z=4

Uroguanylin Doubly Charged Species

23

UroguanylinY = -11732.2+366792*X R^2 = 0.9977 W: 1/X^2

0 100 200 300 400 500 600 700 800 900 1000 1100ng/mL

0

20000000

40000000

60000000

80000000

100000000

120000000

140000000

160000000

180000000

200000000

220000000

240000000

260000000

280000000

300000000

320000000

340000000

360000000

380000000

400000000

Are

aQ Exactive: 100 pg/mL to 10,000 pg/mL – Uroguanylin in Glucagon Peptide Matrix (t-SIM) R2=0.9977, Linear 1/x2

UroguanylinY = -11732.2+366792*X R^2 = 0.9977 W: 1/X^2

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0ng/mL

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

1300000

1400000

1500000

1600000

1700000

1800000

1900000

2000000

Are

a

Linear Dynamic Range 1 x 10(4)

24

Insulin Quantitation Results - Plasma

Nominal Concentration

(ng/mL)Replicate #

Mean Calculated

ConcentrationStdev % CV

0.25 4 0.260 0.0300 11.50.5 4 0.434 0.0501 11.51 4 0.906 0.0540 5.96

2.5 4 2.81 0.0568 2.025 4 4.79 0.112 2.33

10 4 10.3 0.284 2.7725 4 27.9 0.247 0.8950 4 44.0 1.35 3.08100 4 92.8 4.29 4.62250 4 276 9.99 3.62

1000 4 979 36.7 3.75

25

Exendin Quantitation Results - Plasma


(ng/mL)

Replicate #

Mean Calculated


5 4 5.143 0.418 8.1310 4 9.693 0.520 5.3625 4 24.37 1.22 5.0050 4 45.46 0.705 1.55100 4 92.83 13.0 14.0250 4 261.7 5.31 2.03500 4 507.3 2.03 0.40

1000 4 966.0 25.2 2.612500 4 2675.6 60.7 2.275000 4 5205 169 3.2410000 4 9740 150 1.54

26

GLP-1 Quantitation Results - Plasma


(ng/mL)

Replicate #

Mean Calculated


0.1 4 0.099 0.0098 9.910.25 4 0.251 0.0205 8.160.5 4 0.534 0.0337 6.311 4 0.905 0.0226 2.50

2.5 4 2.69 0.0578 2.155 4 5.36 0.126 2.36

10 4 9.99 0.120 1.2025 4 26.6 0.664 2.50250 4 50.6 0.562 1.11100 4 80 0.692 0.86250 4 243 14.2 5.86500 4 462 15.5 3.35

1000 4 860 7.90 0.925000 4 5383 62.8 1.1710000 4 9796 164 1.67

27

PROTEOMICS RESEARCH TO ROUTINE

28

Define the Experiment

Selection of Peptides

Selection of Targeted m/z values

Verification of Targetedm/z values

Optimization of Targetedm/z values

Identify the protein sequence under study

Literature search, in silico digestion, BLAST searchers to ensure sequencespecificity WRT to the background matrix

Sensitivity based on intense fragmentation, selectivity WRT background

Comparison of experimental LC-MS and MS/MS parameters to references(incorporation of recombinant protein analysis and/or heavy labeled synthetic peptides

Keeping only the best peptides/product ions, collision energies, scheduled time windows

Workflow Steps

29

Workflow for Targeted Protein Quantitation Development

Protein(s)

Digest

Separation HPLC

Heavy Labeled Peptides

Heavy Labeled Protein

PRTC Kit

Time →

2. MS(full spectra)

1. Ionization(electrospray)

y??1024.58

b??949.42

y??290.27

y??-NH?860.53

b??648.28

b??247.07

y??662.37

y?²?439.35

y?²?-H?O, y?²?-NH?430.38

y??476.34

[M+2H]²?-H?O, [M+2H]²?-NH?553.27

y??749.47

y??877.53

200 300 400 500 600 700 800 900 1000 1100m/z

0

1000

2000

3000

4000

Inte

nsity

[cou

nts ]

Quantification

Peptide sequenceverification

3. MSPre-cursor selection

4. Fragmentation(CID, HCD, ETD)

5. MSn(Product Ion spectra)

ReferenceDatabase

m/z

MS

Sample Preparation Mass Spectrometry Data Processing

m/z

MS/MS

Domon and Aebersold Nature Biotechnology 2010, 7(28), 710-721

30

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44Time (min)

20.79

20.79

Target Verification Using Heavy Labeled Peptides

4 6 80

1020

30

4050

60

7080

90

10001020

30

4050

60

7080

90

100

NL: 1.71E5

NL: 5.33E6

Rel

ativ

e A

bund

ance

Rel

ativ

e A

bund

ance

561 562

LC

563 564 565 566 567 568 569 570 571 572 573m/z

VFQSWWDR

VFQSWWDR

Rel

ativ

e A

bund

ance

567.2720

567.7727

568.2739

568.7753562.2684

562.7704

MS

200 300 400 500 600 700 800 900 1000

877.46

749.45

553.21

476.22430.39

662.33544.29247.12860.61219.15 375.18 512.81 949.36290.14 648.37

759.33 887.38

558.52

486.35444.33

672.40435.22247.09

219.12 517.79 648.31 870.44375.19300.17

VFQSWWDR

VFQSWWDR

MS/MS

VFQSWWDRm/z 567.2720

VFQSWWDRm/z 562.284

31

HeavyPeptideTM AQUATM Standards

32

HeavyPeptideTM AQUATM Benefits

• HeavyPeptide™ AQUA™ products enable absolute quantification of all proteins in a sample.

• The HeavyPeptide AQUA kits can now be prepared with covalent modifications, such as phosphorylation, which are chemically identical to naturally occurring post-translational modifications (PTMs). As a result, the HeavyPeptide AQUA kits are an extremely cost-effective solution, enabling researchers to identify and quantify peptides of interest much faster, with significantly increased precision.

• This answers the need for relative and absolute quantification of the expression levels for all proteins in complex samples. This is essential since PTMs significantly increase the size of proteomes over their corresponding genomes.

• The HeavyPeptide AQUA product range produces a clear and consistent gain in efficiency, transparency and reproducibility of experiments.

33

Experimental

Control

H2O2 treated (40 hrs)

pH 8.0 treated (40 hrs)

Purified Protein Stock Solution

Treatment Digestion PRTC Kit

0 10 20 30 40 50 60 70 80 90 100 110120 130Time (min)

020406080100020406080100

Rel

ativ

e Ab

unda

nce

020406080100

b₁₄⁺-NH₃, y₁₄⁺1580.73

b₃⁺306.13

b₁₄⁺1597.76

y₃⁺-NH₃444.35

b₅⁺-H₂O462.23

y₂⁺333.30

y₃⁺461.28

b₁₃⁺1411.69

y₁₂⁺1438.74

y₁₁²⁺-H₂O, y₁₁²⁺-NH₃667.34

y₁₁⁺1351.66

b₈⁺843.46

y₁₂²⁺720.39

y₁₀⁺1264.62

b₇⁺756.43

y₇⁺901.54

y₉⁺1101.60

b₉⁺-H₂O938.52

b₁₀⁺-H₂O1039.54

y₈⁺988.54

y₆⁺788.43

y₅⁺687.38

400 600 800 1000 1200 1400 1600

m/z

0

20

40

60

80

100

Inte

nsity

[co

un

ts] (1

0^3

)

Extracted from: C:\Scott Peterman\Backup\Marketing_Monthly_Updates\2011_Marketing_Plan\Thermo_Instruments\Seed_Unit_Requests\Amgen\Control_trainer_180grad_inj1.raw #6273 RT: 88.55 ITMS, CID, z=+2, Mono m/z=872.43262 Da, MH+=1743.85796 Da, Match Tol.=0.8 Da

b₅⁺627.40

a₇⁺-NH₃782.24

y₆⁺620.40

b₆⁺726.32

b₇⁺-H₂O809.40

y₁₂⁺1375.65

y₇⁺749.40

b₁₁⁺1237.40

y₁₃⁺1462.69

b₉⁺-H₂O1075.47

y₈⁺886.46

b₈⁺-H₂O946.40

b₁₁⁺-H₂O1219.45

b₈⁺964.44

b₁₄⁺-H₂O1548.63

b₁₂⁺-H₂O1320.52

y₉⁺987.59

b₁₀⁺1150.41

b₁₃⁺-H₂O1419.59

b₉⁺1093.47

b₁₄⁺1566.60

[M+1H]⁺-H₂O1694.69

600 800 1000 1200 1400 1600

m/z

0

5

10

15

20

25

30

Inte

nsity

[co

un

ts] (1

0^3

)


y₃⁺376.26

b₅⁺560.39

a₃⁺-NH₃295.32

[M+2H]²⁺525.22

b₄⁺503.28

b₃⁺340.31

b₂⁺227.16

y₇²⁺412.34

y₅⁺547.22

y₇⁺823.48

y₆⁺710.39

200 300 400 500 600 700 800 900 1000

m/z

0

10

20

30

40

50

60

70

80

90

Inte

nsi

ty [c

ou

nts

] (1

0^3

)


Sequence for Spectral Libraries

Sequence coverage, AUC, PTMs, %CVs, RT confirmation

34

• What’s New?• Peptide Retention Time Calibration Kit• Pinpoint 1.2 Software for Targeted Protein Quan

• Application:• Quickly assess and optimize chromatography and MS instrument

performance• Predict peptide retention times using calculated hydrophobicity

factors • Predict peptide elution across multiple instrument platforms • Improve quantification and increase multiplexing with optimized

scheduled SRM windows • Incorporation of the PRTC kit provides a system QC,

normalization, and RT correlation across experiments and instrumental platforms.

• Key features• High-purity 15 synthetic heavy peptides mixed at equimolar ratio• Elutes across entire chromatographic gradient• Fully automated using Pinpoint 1.2 QC page

Pierce Peptide Retention Time Calibration Kit + Pinpoint 1.2 Software

Pinpoint

35

Peptide Retention Time Calibration (PRTC) Kit (Heavy Peptides)

# Sequence Observed Mass( Z=2)

HydrophobicityFactor (HF)

1 SSAAPPPPPR 493.7 7.5681

2 GISNEGQNASIK 613.3 15.50003

3 HVLTSIGEK 496.3 15.52207

4 DIPVPKPK 451.3 17.65144

5 IGDYAGIK 422.7 19.15385

6 TASEFDSAIAQDK 695.8 25.8834

7 SAAGAFGPELSR 586.8 25.23967

8 ELGQSGVDTYLQTK 773.9 28.36797

9 GLILVGGYGTR 558.3 32.17702

10 GILFVGSGVSGGEEGAR 801.4 34.51977

11 SFANQPLEVVYSK 745.4 34.96488

12 LTILEELR 498.8 37.30326

13 NGFILDGFPR 573.3 40.41916

14 ELASGLSFPVGFK 680.4 41.18506

15 LSSEAPALFQFDLK 787.4 46.66305

36

Retention Time Analysis – Comparison with Internal Standards

2.14591x + 7.46657 R2 = 0.95902

1.91004x + 11.44465 R2 = 0.97641

37

Conclusions

• Quanfirmation enabled characterization and quantification were performed in 1 experiment

• Separating quantification (MS-level) and qualitative enables reprocessing data for targeted peptide expansion

• Introduction of PRTC kit enabled method reproducibility for the AS, LC, and MS methods

• PRTC kit provides direct relationship of calculated hydrophobicity factors to measured retention times and scalability

• Absolute and/or relative quantitation using heavy peptides• Entire method is integrated with Proteome Discoverer and Pinpoint

38

Acknowledgments

• Kevin Cook• Zhiqi Hao• Scott Peterman

Thank You

research to routine workflows for large and small molecules using...

Documents